PROJECT SUMMARY Nexilin or NEXN (encoded by NEXN) is a recently discovered Z-disc protein. NEXN is highly abundant in cardiac muscle. Multiple mutations in NEXN have been associated with cardiomyopathies, highlighting its importance for cardiac function. A single amino acid substitution of arginine 279 to cysteine (R279C) in NEXN was identified as causing familial hypertrophic cardiomyopathy (HCM), and a 3-base pair (bp) deletion (1948? 1950del) leading to loss of glycine at position 650 (G650del) was found to be highly associated with dilated cardiomyopathy (DCM). Loss of NEXN in zebrafish results in perturbed Z-disk stability and heart failure. Interestingly, myocardial biopsies showed that NEXN mutation carriers exhibit the same cardiac Z-disk pathology as observed in NEXN deficient and mutant zebrafish. Furthermore, global loss of NEXN in mice has been reported to cause rapidly progressive cardiomyopathy with left ventricular dilation, wall thinning, and decreased cardiac function, resulting in lethality shortly after birth. Premature lethality of these mice has prevented study of the role of NEXN in adult heart function. In addition, little is known as to the specific role of NEXN in cardiomyocytes, or mechanisms by which global loss of NEXN in mice results in rapidly progressive cardiomyopathy. Furthermore, mechanisms by which the NEXN R279C and G650del mutations lead to the progression of cardiomyopathy remain to be addressed. To address these questions, we have successfully generated a floxed NEXN mouse line and will use it to generate NEXN cardiac-specific knockout (KO) mice both during developmental and at adult stages. In addition, we have generated novel R274C (equivalent to human R279C) and G645del (equivalent to human G650del) knock-in mouse models to characterize the role of the human NEXN R279C and G650del mutations in cardiomyopathy. Our preliminary characterization of global NEXN KO mice also suggests that NEXN plays an important role in regulating cardiomyocyte cell cycle activity. The foregoing scientific premise leads us to the hypothesis that NEXN plays an essential role in regulating cardiomyocyte cell cycle activity as well as maintaining the structural integrity of the Z-line during the stress of muscle contraction, and that R279C and G650del mutations in NEXN impair specific aspects of NEXN function to lead to HCM and DCM, respectively. Our Specific Aims are: 1. To characterize the role of NEXN in the developing and adult myocardium by analyzing NEXN global and cardiomyocyte-specific knockout mice both during development and at adult stages for cardiac function, cardiomyocyte cell cycle activity, sarcomere integrity, and the progression of cardiomyopathy; and 2. To elucidate mechanisms underlying cardiomyopathy consequent to R279C and G650del mutations of NEXN by analysis of R274C or G645del mutant mice and human embryonic stem cell-derived cardiomyocytes containing NEXN R279C or G650del mutations.